title: Solid-support fluorescent derivatization of picomoles of protein at low concentration with FITC.
abstract: A new method based on solid-support reaction is described to realize fluorescent derivatization of proteins at concentrations as low as 10(-8)M. A simple, low-cost homemade capillary C18 cartridge was fabricated as the solid-support reactor. Using bovine serum albumin (BSA) as a test protein, we demonstrated that the protein can be captured by this reactor and then labeled by fluorescein isothiocyanate (FITC, isomer I) on solid-support. Unwanted fluorescent intruder (excrescent FITC and products of secondary reactions) were removed from target easily. The analysis by nano-HPLC with laser-induced fluorescence (LIF) detection was described. The effect of reaction conditions on the derivatization has been evaluated and discussed. The use of the solid-support reactor allows easy handling of as little as 8.5 pmol of BSA. A fraction from weak anion-exchange chromatography (WAX) of human liver extract was used as an illustrative example of application to real samples.
authors.affiliation: Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Tao
authors.forename: Ming-Xia
authors.initials: MX
authors.lastname: Gao
authors.forename: Guang-Feng
authors.initials: GF
authors.lastname: Hong
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.forename: Xiang-Min
authors.initials: XM
authors.lastname: Zhang
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D036103:Nanotechnology; D012710:Serum Albumin, Bovine / Q000737:chemistry*
chemical_list: D012710:Serum Albumin, Bovine; D016650:Fluorescein-5-isothiocyanate